Francisella tularensis is a facultative intracellular bacterial pathogen that causes a spectrum of diseases collectively called tularemia. Two subspecies, subsp. tularensis (type A) and subsp. holarctica (type B) can cause severe disease in humans. In particular, inhalation of small numbers of type A F. tularensis has a mortality rate of 30-60% if left untreated (Sjostedt, 2007). In contrast, type A F. tularensis infections initiated by non-respiratory routes are far less lethal, and type B infections initiated by any route can cause debilitating, but non-life-threatening disease in humans.
An empirically attenuated type B strain of F. tularensis developed more than 50 years ago, F. tularensis live vaccine strain (LVS), has been used to protect against exposure to virulent type A strains of the pathogen. In formal testing using human volunteers, LVS was shown to impart complete protection against transdermal challenge with the type A strain SCHU S4, though it afforded lesser protection against an aerosol challenge (Saslaw et al 1961a, 1961b). It is the sole vaccine to have been formally shown to possess these properties. Due to safety concerns, it has never been fully licensed by the U.S. Food and Drug Administration (FDA).
Genomic sequencing of clinical type A and type B strains of F. tularensis as well as LVS allowed identification of the genetic modifications in the vaccine strain. Much of the attenuation of LVS versus clinical type B strains appears to be due to defects in a pilus gene, pilA, and a gene (FTT0918) of unknown function (Salomonsson et al. 2009). LVS also contains multiple other minor mutations that, separately or collectively, contribute to its attenuation.
LVS is known to elicit both an antibody response and a CD4+ and CD8+ T-cell response to several F. tularensis proteins. Experiments in mice indicate that the ability of LVS to elicit CD4+ and CD8+ T-cells secreting interferon gamma accounts for its efficacy against type A strains (Conlan et al. 2005; Wu et al. 2005). However, C57BL/6 mice that produce both antibodies and gamma interferon-secreting T-cells (Woolard et al. 2008; Twine et al 2006) following vaccination with LVS are not protected from challenge with type A bacteria (Chen et al. 2003, Wu et al 2005; Green, et al. 2005). Ignorance of the mechanism of protection of LVS and of the relative contributions of each of its mutations to its overall attenuation are major barriers to its full licensure by the U.S. FDA. The antigens of LVS that are responsible for eliciting protective immunity are unknown; additionally, because LVS is a vaccine generated from a type B strain, virulence factors and other macromolecules unique to type A strains are missing. These facts render difficult the task of designing specific antigen-based vaccines.
In recent years, various mutagenesis strategies have been used to identify virulence factors of Francisella that could be disrupted to produce novel live vaccine strains. Much of this work has been performed using LVS or F. novicida, a related subspecies of the pathogen that is only virulent for immunosuppressed humans. This approach relies on two critical assumptions regarding the use of LVS or F. novicida as surrogate clinical strains: 1) genes that are required for virulence of LVS or F. novicida predict virulence genes for clinical isolates; and 2) vaccines that protect against LVS or F. novicida will predictably protect against clinical strains.
However, LVS is already approximately 1,000,000-fold less virulent than clinical type A and B strains of the pathogen; thus, inhibiting the expression of any other virulence genes in LVS will only have an incremental effect on virulence. This renders impossible the prediction of effect any such mutation would have on a fully-virulent strain of F. tularensis in the absence of the innate mutations of LVS. Furthermore, it has been shown that mutant strains of LVS or F. novicida are able to protect mice against challenge with the homologous wild-type strain, but not against challenge with fully virulent type A bacteria (Quarry et al 2007; Sebastian et al 2007). Additionally, antibodies against surface lipopolysaccharides protect against F. novicida and type B strains, but fail to protect against Type A bacteria (Conlan et al 2002; Fulop et al 2001; Thomas et al. 2007). Finally, there appears to be no correlation between protection and vaccine-elicited antibody titre (Saslaw and Carhart 1961).
Furthermore, vaccines composed of killed cells and fractions thereof are sub-optimally effective against F. tularensis because such preparations fail to generate robust and prolonged protective cell-mediated immunity. Hence, there is currently no FDA-approved vaccine for general use that can provide prophylactic protection against respiratory tularaemia.